Lyme disease, due to infection with the Ixodes tick-transmitted spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States, with over 20,000 cases reported to the CDC annually. After deposition in the skin, spirochetes can disseminate and incite clinically apparent disease in several sites, especially the skin, heart, joints and nervous system. While antibiotics are successful in achieving clinical cure in the majority of patients with early Lyme disease, those with disseminated infection or in whom treatment is delayed can experience lingering signs and symptoms of uncertain etiology. The timely and accurate diagnosis of Lyme disease is essential for optimizing the response to therapy and for preventing long-term sequellae of the disease. Serologic tests (ELISA and immunoblot) that detect antibodies against B. burgdorferi are currently the most sensitive and widely available laboratory tests for Lyme disease. These tests utilize whole-cell lysates of cultured laboratory strains of B. burgdorferi as a source of antigens, and therefore do not detect antibodies to B. burgdorferi proteins expressed exclusively in vivo. The recommended two-tiered approach in which a positive or equivocal ELISA is confirmed by immunoblot has only ~30% sensitivity in early stages of infection, and specificity is reduced due to cross-reactivity of B. burgdorferi antigens with those of other infectious agents. Moreover, the current Lyme ELISA and immunoblot tests do not distinguish previous exposure to B. burgdorferi from active infection, and no serologic test to date can be used to assess response to therapy. This Phase I proposal seeks to improve upon the currently available serologic tests for Lyme disease through a proteomic approach that will a) identify immunodominant B. burgdorferi proteins expressed in vivo, and b) evaluate their utility as diagnostic antigens at various stages of B. burgdorferi infection. The key to this approach is the use of a mutant mouse strain in which spirochetes can achieve high pathogen burden in tissues under conditions in which antibody-mediated selection pressure on the organism is preserved. These novel mutant mice will be used as a tissue source of host-adapted B. burgdorferi and its in vivo expressed antigens. 2D-differential fluorescence gel electrophoresis, immunoblot, and MALDI-MS/MS will be used to identify immunodominant B. burgdorferi antigens expressed exclusively in mammalian tissues in early and late stage vector-borne infection. Candidate diagnostic antigens will be produced as recombinant proteins and tested for reactivity with a panel of sera representative of various stages B. burgdorferi infection. The results of this Phase I study will set the stage for the development of new sensitive and specific serologic tests for Lyme disease that more accurately reflect the B. burgdorferi antigens inciting immune responses in the infected host. [unreadable] [unreadable] Lyme disease, due to infection with the spirochete Borrelia burgdorferi, can cause a multi system illness involving the skin, heart, joints and nervous system. Timely diagnosis of the disease is critical for optimizing treatment and for the prevention of long-term sequellae. This proposal will use a proteomics approach to identify novel immunodominant B. burgdorferi proteins that are predominantly expressed within the mammalian host. Identification and characterization of these proteins will set the stage for the development of improved serologic tests for Lyme disease in both early and late stage infection. [unreadable] [unreadable] [unreadable] [unreadable]